To date there are a lot of drugs and vaccines for various infectious diseases of cattle, but in some cases are not as effective if the disease is in an advanced stage. Millions of dollars are spent on research, which is mostly aimed at finding an effective treatment, downplaying the timely detection of the disease through a good method, which in many cases can lead to appropriate treatment with excellent results and offer a control on these diseases (1).
Parallel to that investment, statistics showing areas in which diseases such as bovine tuberculosis and brucellosis have not been eradicated have not decreased significantly, representing a major public health problem because they are considered zoonotic diseases (OIE, 2004) (NOM-031-ZOO-1995). (2,3)
In Mexico, the 83.11% of the country is in the process of eradication of bovine tuberculosis with a prevalence less than 0.50%, the rest of the country has a prevalence of less than 2.05%. As for brucellosis, it has been eradicated only in the state of Sonora, but in the rest of the country it has not been possible (SENASICA, 2011). Upon controlling and eradicating bovine tuberculosis a potential source for human infection will be eliminated and this has been demonstrated in several countries through campaigns of prevention, control and eradication of tuberculosis (NOM-031-ZOO-1995).
Each year, hundreds of millions of animals are slaughtered in the world because they are infected by these pathogens, causing great economic losses to livestock (NOM-031-ZOO-1995). The existing problem for both is the lack of early detection, which would allow a best control over the disease. This lack of detection in the case of brucellosis is because an unequivocal diagnosis can only be made by isolation and identification of Brucella, but in situations where bacteriological analysis is not possible, the diagnosis can be based on serological methods. There is no single test that allows to identify the disease, and normally a combination of growth characteristics and bacteriological and serological methods is required to determine whether the animal is infected (OIE, 2004). In the case of bovine tuberculosis, there is no clinical evidence of the disease in cattle until they have developed very extensive lesions. The measures implemented by the government are reflected in the rigorous application of the tuberculin test and the selection of cattle with positive reaction, measures that have not been very effective to date, so in general have been unsuccessful (OIE, 2004).
Moreover, other factors also influence the non timely detection of infectious diseases in cattle, such as the level of development, the quality of health systems, and access to accurate and cost-effective means of diagnosis in each country. The majority of developing countries, which lead the statistics of these diseases, have this problem. However, although some countries with economic potential have new technologies, including some commercially available for detection of the disease, they are expensive and inaccessible for most of livestock farmers. Also, diagnosis is usually reached when the damage is irreversible (OIE, 2004). Therefore, the need to create products that are sensitive to detect diseases even without clinical signs and at any stage of the disease is a priority. In that sense, the diagnostic methods used until today can be effective, but require careful sample preparation, trained staff, and especially are time consuming. Other methods that can be considered fast are not very accurate and the results are not reliable.
The classic test to detect bovine tuberculosis is the tuberculin, one of the problems of this test is that it does not detect animals without delayed-type hypersensitivity (DTH) to Mycobacterium bovis, termed anergic, either with a disseminated infection or a recent infection, because in such cases the cellular immunity is depressed or in development (Ritacco, 1991). On the other hand, they are laboratory tests of blood, such as lymphocyte proliferation test, the interferon gamma and enzyme immunoassay. The logistics and performing of these tests in the laboratory may be a limiting factor, thus more comparative studies of these new tests and skin tests in different field conditions are needed (OIE, 2004). Another disadvantage of tuberculin is that has a high number of false positives, because it is used a protein extract that induces cross-reactivity with non-pathogenic mycobacteria.
Works published such as Patarroyo et al. support the use of Mycobacterium antigens like ESAT-6 for tuberculosis detection, as it has been shown that this antigen can detect active or latent Mycobacterium. In this sense, the document US20060024332A1, teaches the use of a fusion protein, consisting of recombinant antigens like ESAT-6 to stimulate leucocytes and producing gamma interferon. It is considered a specific and sensitive method, but in economic terms the cost is very high and impractical. Other published works, such as US20100166786A1, US20100015096A1, US006982085B2 and US7261897B2 show the use of fusion proteins, consisting of at least two antigens of tuberculosis, and although they have the function of detecting tuberculosis, its main objective is directed to be used as preventive vaccines and also require specialized equipment and that raises the cost of the product, besides being directed to humans so far.
The detection of brucellosis is made by tests with buffered Brucella antigen (rose bengal) and the buffered plate agglutination test, as well as complement fixation test, enzyme-linked immunosorbent assay (ELISA) or fluorescence polarization assay. Despite they can be used to screen herds and individual animals, is a fact that no single serological test is suitable for each and every one of the epidemiological situations. Therefore, the reactivity of the samples that are positive in screening tests should be confirmed using a confirmatory strategy established. The indirect ELISA or the milk ring test, made with whole milk samples, are effective for analyzing and controlling brucellosis in dairy cows, but the milk ring test is less reliable in large herds. Another immunological test is the Brucellin skin test, which is used in analysis or as a confirmatory test in positive herds (OIE, 2004).
To a large extent several diagnostic tests for tuberculosis and brucellosis have been disclosed, which characterize by possessing antigens characteristic of the disease, or antigens that may be merged with another protein that can detect the disease, despite they mainly are used as vaccines. Although some have high sensitivity and specificity according to the investigations reported, U.S. Pat. No. 7,632,646B1 or US20100166786A1 have the disadvantages of being uneconomical, impractical and most can hardly be used in non-urban areas, causing a large consumption of transportation time, equipment and personnel training.
In order to provide a product that suppresses these drawbacks also have been created diagnostic tests that involve fusion or recombinant proteins, which are characterized by having a protein that recognizes proteins of bovine erythrocytes and other protein that detects antigens or antibodies characteristic of an infectious disease, producing a hemagglutination reaction (Shohet et al. 1985). In this sense, for some time now are available works such as WO9324630 and U.S. Pat. No. 7,585,508B1 that are clear examples of this type of recombinant proteins used in erythrocyte protein recognition by one of its proteins. In the first study, a malaria peptide is identified, and in the second a monoclonal antibody from a hybridoma; the latter shows that by merging with disease-specific antigens they produce a hemagglutination reaction, with a sensitivity of 100%. However, the use of larger volumes and concentrations is mentioned for recognizing erythrocyte protein than the protein that is the object of this invention, which has a sensitivity in the range of 90-100%, with a single addition of protein, which makes it more convenient, fast and efficient than previous ones.
This invention proposes a protein from shark Heterodontus francisci, which recognizes bovine erythrocyte membrane proteins and furthermore can be fused with proteins such as ESAT-6, and infectious diseases, such as tuberculosis and brucellosis. The recombinant protein can detect the disease, recognizing bovine erythrocytes and antibodies produced by the disease through a hemagglutination reaction.
Therefore, the invention disclosed herein is a good alternative to detect antibodies that may be present in infectious diseases of livestock, which will make the process very fast and that can be detected in cattle that present no symptoms and with infection in active state. Another advantage of this invention to be protected is that the costs of training personnel and logistical issues can be eliminated. Also is not limited to required concentrations of the bacillus to get a result, what happens to other diagnostic tests that are also impractical in rural areas.